The molecular motor kinesin-1 performs a large number of transport tasks, and the regulatory mechanisms governing those processes are critical. Mis-regulation of kinesin-1 or mis-localization of kinesin-1 cargoes may be implicated in several diseases such as Parkinsons disease, neurofibromatosis, schizophrenia, and Charcot-Marie-Tooth disease. Kinesin-1s motile mechanism is well understood, and we now also know that kinesin-1s C-terminal tail interacts directly with and inhibits the heads when the motor is not needed for cargo transport. However, we do not know how kinesin-1 regulators initiate or stop cargo movement. The tail is certainly involved, as it binds to heads, microtubules, and several distinct kinesin-1 activators that function in different transport complexes. Separate from the tail, the Miro protein has a direct, Ca2+-dependent interaction with kinesin-1s enzymatic head domains, and Miro is required for Ca2+-dependent suppression of mitochondrial motility. We hypothesize that the tail is an intrinsically disordered domain, having structural flexibility that facilitates multiple binding partner interactions involved in kinesin-1 auto-inhibition and/or activation, while Miro has a distinct mechanism, directly inhibiting the enzymatic mechanism of kinesin-1 heads to suppress mitochondrial movement. To address this hypothesis, we will first gain detailed information in vitro about the structure of the kinesin-1 tail and its interactions with binding partners, by NMR and EPR spectroscopy. We will determine whether Miro is a direct, Ca2+-switchable inhibitor of kinesin-1s enzymatic activity, assess its effects on kinesin-1 mechanism using EPR, and map its interaction with kinesin-1 heads by cross-linking. After obtaining this structural and mechanistic information on both the tails and Miro, we will determine whether and how they influence mitochondrial movement by controlling kinesin-1 in vivo, by imaging mitochondria in live Drosophila S2 cells. These Aims together will provide an exciting new bridge between in vitro biophysical and cell biological work on molecular motor transport mechanisms. Furthermore, as Miro and other kinesin-1 regulators have been implicated in several neurological diseases, our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.